Open AccessMonoclonal Antibodies Against Two Electron Reduced Riboflavin and a Quantification of Affinity Constants for this Oxygen‐Sensitive Molecule
Author(s) -
Bruggeman Yvonne E.,
Schoenmakers Ronald G.,
Schots Arjen,
Pap Everard H. W.,
Hoek Arie,
Visser Antonie J. W. G.,
Hilhorst Riet
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.245_c.x
Subject(s) - flavin group , riboflavin , chemistry , flavin adenine dinucleotide , flavoprotein , monoclonal antibody , fluorescence , photochemistry , antibody , biochemistry , enzyme , cofactor , biology , physics , quantum mechanics , immunology
In order to create a protein environment that binds preferentially to the two‐electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two‐electron‐reduced flavin using standard techniques. Because of its sensitivity, time‐resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG 1 and an IgM, to reduced riboflavin (1,5‐dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG 1 is more than 80 times larger for 1,5‐dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5‐dihydroriboflavin bound to IgG 1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5‐dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.